Profiling Glycosyltransferase Activities on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using peptide arrays and mass spectrometry

使用肽阵列和质谱分析乳腺上皮细胞上皮-间质转化中的糖基转移酶活性

• 批准号: 9249389
• 负责人: Jose-Marc Techner
• 金额: $ 4.9万
• 依托单位: NORTHWESTERN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9249389
• 关键词: Address; Biological Assay; Biological Markers; Biotechnology; Breast; Breast Cancer cell line; Breast Epithelial Cells; Cell Line; Cell model; Cells; Chemistry; Clinical; Communities; Complex; Data; Detection; Development; Developmental Process; Effectiveness; Enzymes; Epithelial; Epithelial Cells; Fetal Tissues; Fibronectins; GALNT3 gene; Galactosyltransferases; Generations; Genetic; Glycobiology; Glycoproteins; Goals; Lead; Lung; MALDI-TOF Mass Spectrometry; MCF10A cells; MDA MB 231; Malignant - descriptor; Malignant Neoplasms; Mammary Gland Parenchyma; Mammary gland; Maps; Mass Spectrum Analysis; Measures; Mediating; Mesenchymal; Metastatic breast cancer; Methods; Negativism; Neoplasm Metastasis; Outcome; Peptide Library; Peptides; Phenotype; Post-Translational Protein Processing; Process; Prostate; Protein Glycosylation; Protein Isoforms; Proteomics; Research; Role; Sampling; Small Interfering RNA; Spectrometry; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; System; Techniques; Technology; Testing; Time; Tissues; Transforming Growth Factor beta; Transitional Cell Carcinoma; Work; base; cancer cell; cancer subtypes; enzyme activity; enzyme substrate; epithelial to mesenchymal transition; experimental study; genome sequencing; glycosylation; glycosyltransferase; high throughput screening; improved; innovative technologies; knock-down; loss of function; malignant breast neoplasm; monolayer; novel; novel marker; overexpression; public health relevance; sugar; therapeutic target; treatment response; triple-negative invasive breast carcinoma; tumor DNA; whole genome

 DESCRIPTION (provided by applicant): Profiling Glycosyltransferase Activity on the Epithelial-Mesenchymal Transition in Breast Epithelial Cells using Peptide Asrrays and Mass Spectrometry High-throughput biotechnology has enabled discovery of genetic and proteomic profiles that identify cancer subtypes with distinct phenotypes. However, similar efforts to profil based on the posttranslational modifications mediated by glycosyltransferases have lagged due to lack of efficient high-throughput assays. Such enzymatic profiling technology is needed because these enzymes exert dramatic effects on cell phenotype. For example, fibronectin glycosylation by GalNac-T3 and GalNac-T6 in prostate cells epithelial cells is essential for the TGF-beta induced epithelial-mesenchymal transition (EMT), a developmental process that cancer cells hijack to enable metastasis. This example is particularly relevant to breast cancer because this form of fibronectin, onfFN, is upregulated in breast cancer tissue and not present in normal mammary tissue. Moreover, overexpression of GalNac-T6 induce an EMT-like phenotype in MCF10A, an immortalized breast epithelial cell line. Despite these evidence it remains unclear if GalNAc-T3 and GalNAc-T6 regulate EMT in breast tissue by a similar mechanism as that in prostate cells. Therefore my hypothesis that GalNAc-T3 and GalNAc-T6 promote EMT in breast cells via glycosylation of fibronectin and that their activity can serve as novel biomarkers of EMT. This proposal seeks to investigate the role of GalNAc-T3/-T6 on TGF-beta EMT in breast cells and use an innovative technology to profile their activity directly from cell lysate, known as SAMDI mass spectrometry. This method uses peptide substrate arrays to profile enzyme activities in a sample and has demonstrated effectiveness for studying several enzymatic chemistries, including glycosylation. Preliminary data further demonstrate that SAMDI profiling is feasible via detection endogenous galactosyltransferase activity in the triple-negativ breast cancer cell line, MDA-MB-231. The first aim will entail synthesis of a peptide library that can distinguish activities between GalNAc-T3 and GalNAc-T6 and then test by SAMDI whether GalNAc-T3 and GalNac-T6 activities can be used to profile EMT. In the second aim, I will investigate the extent to which GalNAc-T3 and -T6 regulate EMT in breast epithelial cells. Using overexpression and knockdown studies, I will test whether EMT is dependent on the expression of GalNAc-T3/-T6 and whether EMT is dependent on the presence of onfFN. This proposal aims to address two critical needs: fill a gap in available technology relevant to the field of glycobiology as a whole as well as provide fundamental information important to the breast cancer community.

 描述（由申请人提供）：使用肽阵列和质谱分析乳腺上皮细胞中上皮-间质转化的糖基转移酶活性高通量生物技术已经能够发现识别具有不同表型的癌症亚型的遗传和蛋白质组谱。基于糖基转移酶介导的翻译后修饰的概况已经滞后，因为缺乏有效的高通量分析技术是必要的，因为这些酶对细胞表型产生显着影响，例如，前列腺细胞上皮细胞中 GalNac-T3 和 GalNac-T6 的纤连蛋白糖基化对于 TGF-β 诱导至关重要。上皮间质转化（EMT），癌细胞劫持以实现转移的一种发育过程，这个例子与乳腺癌特别相关，因为这种形式的转移。纤连蛋白 onfFN 在乳腺癌组织中表达上调，但在正常乳腺组织中不存在。此外，GalNac-T6 的过度表达会在永生化乳腺上皮细胞系 MCF10A 中诱导 EMT 样表型，尽管有这些证据，但仍不清楚 GalNac-T6 是否存在。 T3 和 GalNAc-T6 通过与前列腺细胞类似的机制调节乳腺组织中的 EMT，因此我假设 GalNAc-T3 和 GalNAc-T6 促进 EMT。该提案旨在研究 GalNAc-T3/-T6 对乳腺细胞中 TGF-β EMT 的作用，并使用创新技术来分析其活性。直接从细胞裂解物中检测活性，称为 SAMDI 质谱法。该方法使用肽底物阵列来分析样品中的酶活性，并已证明可有效研究多种酶化学，包括。初步数据进一步表明，通过检测三阴性乳腺癌细胞系 MDA-MB-231 中的内源半乳糖基转移酶活性，可以进行 SAMDI 分析，第一个目标是合成能够区分 GalNAc-T3 活性的肽库。和 GalNAc-T6，然后通过 SAMDI 测试 GalNAc-T3 和 GalNac-T6 活性是否可用于分析 EMT。我将研究 GalNAc-T3 和 -T6 在乳腺上皮细胞中调节 EMT 的程度，通过过度表达和敲低研究，我将测试 EMT 是否依赖于 GalNAc-T3/-T6 的表达以及 EMT 是否依赖于 GalNAc-T3/-T6 的表达。该提案旨在满足两个关键需求：填补整个糖生物学领域现有技术的空白，并提供对乳腺癌界重要的基本信息。